9007-43-6

Brown-red crystalline powder

A component of the electron transport chain in mitochondria, cytochrome c is assumed to be the functional complex utilized in low-level laser therapy (LLLT), which increases the metabolic activity and frees up more energy for the cells to repair the tissue.It shows peroxidase activity by oxidation of various electron donors such as 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2-keto-4-thiomethyl butyric acid and 4-aminoantipyrine. It is used as an important mediator in apoptotic pathways.

cytochrome: Any of a group ofproteins, each with an iron-containinghaem group, that form part ofthe electron transport chain in mitochondriaand chloroplasts. Electronsare transferred by reversiblechanges in the iron atom betweenthe reduced Fe(II) and oxidized Fe(III)states.

Cytochrome c is involved in the mitochondrial electron-transfer chain and accepts an electron from cytochrome c1 and transfers it to cytochrome c oxidase. This electron is subsequently used in the reduction of oxygen, where four electrons are needed. This means that actually four cytochrome c transfer an electron to cytochrome c oxidase, where one molecule of O₂ is converted to two molecules of water.

The ready interconversion of cytochrome c between ferrous and ferric states makes it an efficient biological electron carrier. It plays a vital role in cellular oxidations in both plants and animals. Generally regarded as a universal link in the respiratory chain, it forms the essential electron-bridge between the respirable substrates and oxygen.

Cytochrome c1 is purified by chromatography on CM-cellulose (CM-52 Whatman) [Brautigan et al. Methods Enzymol 53D 131 1978]. It has a high PI (isoelectric point) and has been purified further by adsorption onto an acidic cation exchanger, e.g. Amberlite IRC-50 (polycarboxylic) or in ground form Amberlite XE-40 (100-200 mesh) or Decalso-F (aluminium silicate), where the non-cytochrome protein is not adsorbed and is readily removed. The cytochrome is eluted using a solution containing 0.25g ions/L of a univalent cation at pH 4.7 adsorbed onto the NHsalt of Amberlite IRC-50 at pH 7, washed with H2O and then with 0.12M NH4OAc to remove non-cytochrome protein. When the cytochrome begins to appear in the eluate, then the NH4OAc concentration is increased to 0.25 M. The fractions with ca Fe = 0.465—0.467 are collected, dialysed against H2O and adsorbed onto a small IRC-50 column and eluted with 0.5M NH3, then dialysed and lyophilised. (A second fraction II can be eluted from the first resin with 0.5M NH3 but is discarded). [Keilin & Hartree Biochemical Preparations 1 1 1952, Margoliash Biochemical Preparations 8 33 1957.] Cytochrome c has been recrystallised as follows: The above eluate (ca 100mL) is dialysed against H2O (10 vols) at 4o for 24hours (no more), then passed through an XE-40 column (2 x 1 cm above) which is equilibrated with 0.1M NH4OAc pH 7.0. The column is washed with 0.1% (NH4)2SO4 pH 8.0, and the dark red resin in the upper part of the column is collected and in 0.1% (NH4)2SO4 pH 8.0 is transferred to another column (7mm diameter) and the cytochrome c is eluted with 5% (NH4)2SO4 pH 8.0. More than 98% of the red colour is collected in a volume of ca 4mL in a weighed centrifuge tube. Add a drop of octanol and 0.43g of (NH4)2SO4/g of solution. When the salt has dissolved, ascorbic acid (5mg) is added as well as a few drops of 30% NH3, and it is kept at 10o for 10minutes (turns lighter colour due to reduction). Then add finely powdered (NH4)2SO4 in small portions (stir with a glass rod) until the solution becomes turbid. Stopper the tube tightly, and set aside at 15-25o for 2days while the cytochrome c separates as fine needles or rosettes. Further (NH4)2SO4 (20mg) are added per mL of suspension and kept in the cold for a few days to complete the crystallisation. The crystals are collected by centrifugation (5000xg), suspended in saturated (NH4)2SO4 (pH 8.0 at 10o), then centrifuged again. For recrystallisation the crystals are dissolved in the least volume of H2O, one drop of ammonia and 1 mg of ascorbic acid are added and the above process is repeated. The yield of twice recrystallised cytochrome c from 2Kg of muscle is ca 200 mg, but this varies with the source and freshness of the muscle used. The crystals are stored as a solid after dialysis against 0.08M NaCl or 0.1M sodium buffer and lyophilising, or as a suspension in saturated (NH4)2SO4 at 0o. [Hagihara et al. Biochemical Preparations 6 1 1958.] Purity of cytochrome c: This is checked by the ratio of the absorbance at 500nm (reduced form) to 280nm (oxidised form), i.e. should be between 1.1 and 1.28, although values of up to 1.4 have been obtained for pure preparations. For the preparation of the reduced form see Margoliash Biochemical Preparations 5 33 1957 and Yonetani Biochemical Preparations 11 19 1966. Cytochrome from Rhodospirillum rubrum ( 551 0.967) is purified by chromatography on a column of 270/ CM-Whatman cellulose [Paleus & Tuppy Acta Chem Scand 13 641 1959].